Copper oxide wire particles used to control Haemonchus infections : efficacy in giraffe (Giraffa camelopardalis) at Busch Gardens Tampa and potential mechanism of action

Gastrointestinal nematode (GIN) infections affect production systems and exotic hoofstock in zoos, particularly giraffe (Giraffa camelopardalis). Anthelmintic resistance (AR) prevalence is increasing in production systems and zoos. To combat the AR that compounds GIN problems, alternative control methods are used. One such alternative is copper oxide wire particles (COWP), which control the abomasal nematode Haemonchus contortus. COWP was given to seven adult giraffe at Busch Gardens Tampa, at descending dosages: 25 g, 12.5 g, 6.3 g. Treatment administration time was determined by fecal egg count (FEC) being above 600 eggs per gram (EPG), and therefore varied with each animal. FEC following 25 g COWP treatment significantly (p \u3c 0.05) decreased between Week 0 and Weeks 2-5. FEC following 12.5 g COWP treatment significantly (p \u3c 0.05) decreased between Week 0 and Week 2. FEC following 6.3 g COWP treatment showed no reduction between Week 0 and all PT samples. A 25 g COWP treatment was an acceptable part of a GIN control program for treatment of Haemonchus in giraffe, a 12.5 g COWP treatment was minimally acceptable, and a 6.3 g COWP treatment was ineffective. To determine the mechanism of action for COWP, 11 Suffolk lambs were abomasally fistulated and inoculated with H. contortus larvae. Abomasal contents, nematodes, feces, and blood were collected at 0, 12, 36, 60, and 84 hours post-treatment (HPT). pH was measured and nematodes were viewed using scanning (SEM) and transmission (TEM) electron microscopy and energy dispersive x-ray spectroscopy (EDS), and copper content analysis was performed. FEC and packed cell volume (PCV) were determined for each time period. pH was inconclusive. FEC decreased throughout the study and up to 33 days PT; no statistics were performed due to removal of nematodes. PCV had no substantial change, but the study time period was too short for a significant change. SEM showed no obvious changes between pre- and post-treatment. TEM showed cuticle damage present in PT samples. EDS showed copper present in 10 of 11 spots on PT samples. Copper content of nematodes was maximum at 60 HPT. Evidence suggests COWP has a direct effect on H. contortus

Adaptation of Human Parainfluenza Virus to Airway Epithelium Reveals Fusion Properties Required for Growth in Host Tissue

Paramyxoviruses, a family of RNA enveloped viruses that includes human parainfluenza virus type 3 (HPIV3), cause the majority of childhood croup, bronchiolitis, and pneumonia worldwide. Infection starts with host cell receptor binding and fusion of the viral envelope with the cell membrane at the cell surface. The fusion process requires interaction of the two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). We have previously shown that viruses with an HN/F pair that is highly fusogenic in monolayers of immortalized cells due to mutations in HN’s secondary sialic acid binding site are growth impaired in differentiated human airway epithelium (HAE) cultures and in vivo. Here we have shown that adaptation of HPIV3 to growth in the lung is determined by specific features of HN and F that are different from those required for growth in cultured immortalized cells. An HPIV3 virus bearing a mutated HN (H552Q), which is fit and fusogenic in immortalized cells but unfit for growth in the lung, evolved into a less-fusogenic but viable virus in differentiated human airway epithelium. Stepwise evolution led to a progressive decrease in efficiency of fusion activation by the HN/F pair, with a mutation in F first decreasing the activation of F by HN and a mutation in HN’s secondary sialic acid binding site decreasing fusion activation further and producing a stable virus. Adaptation of HPIV3 to successful growth in HAE is determined by specific features of HN and F that lead to a less easily activated fusion mechanism

Effectiveness of Neuraminidase Inhibitors for Preventing Staff Absenteeism during Pandemic Influenza

Timely and adequate treatment and prophylaxis may reduce absenteeism among healthcare workers during the peak of a pandemic

The Use of a Quantitative Fusion Assay to Evaluate HN–Receptor Interaction for Human Parainfluenza Virus Type 3

AbstractSialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin–neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle—functional interaction between viral glycoproteins and the cell during attachment and fusion—and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-βgal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the β-galactosidase (βgal) produced as a result of this fusion. The analog α-2-S-methyl-5-N-thioacetylneuraminic acid (α-Neu5thioAc2SMe) interfered with fusion, decreasing βgal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN–receptor interaction; 10 mM DANA completely blocked fusion and βgal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog α-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN–receptor binding. Strategies suggested by these findings are now being pursued to obtain information regarding the relative locations of the active sites of HN and to further elucidate the relationship between the receptor-binding and receptor-destroying activities of HN during the viral life cycle. The quantitative assay that we describe is of immediate applicability to large-scale screening for potential inhibitors of HPF3 infection in vivo